Interferon regulatory factor 3 supports Th17 cell differentiation and pathogenicity. (A) Splenocytes cells from naive interferon regulatory factor–knockout (KO, irf3
−/−) and wild-type (WT) mice were activated with anti-CD3/anti-CD28 (1 μg/ml) in non-polarised (no exogenous cytokines), Th1-polarising (interleukin-12 (IL-12)) or Th17-polarising (transforming growth factor β (TGF-β), IL-6, IL-1β and antibody against interferon γ (anti-IFN-γ)) conditions for 3 days. Helper T (Th) cell polarisation was analysed by flow cytometry following restimulation with phorbol 12-myristate 13-acetate/ionomycin/GolgiPlug protein transport inhibitor for the final 4 hours of culture. The percentages of positive gated CD4+ live cells are displayed. (B) Splenocytes were activated in other Th17-polarising conditions as indicated for 3 days, and IL-17 production was measured by enzyme-linked immunosorbent assay (ELISA). (C) WT and irf3
−/− mice were immunised with myelin oligodendrocyte glycoprotein (MOG35–55) in complete Freund’s adjuvant. After 10 days, cells from the spleens and lymph nodes were reactivated with MOG35–55 (25 μg/ml) in the presence of IL-23 (20 ng/ml) for 3 days. Cells were transferred into naive WT or irf3
−/− recipient mice. Mice were scored daily for clinical signs of disease (see Table 2). (D) Spleens and lymph nodes were harvested from WT and irf3
−/− mice that had been immunised with MOG35–55 for 7 days. CD4+ and CD4− populations were immunomagnetically purified and cultured in combinations as indicated. Cultures were reactivated with MOG35–55 (25 μg/ml) in the presence of IL-23 (20 ng/ml), and IL-17 was measured by ELISA (n = 4). Data from one experiment representative of two or three replicate experiments are shown.